Multicomponent or monocomponent vaccine to be used against chagas disease, pharmaceutical compositions containing them, procedure for the obtention of immunogen of said vaccines, and nucleic acid used in said procedure

ABSTRACT

A monocomponent vaccine effective to moderate the clinical consequences of Chages disease, capable of stimulating an immune response against the trans-sialidase virulence factor of the Trypanosoma cruzi parasite. The vaccine active ingredient is an immunogenic component with a polynucleotide encoding one or more polypeptide(s) which includes a C-terminal region composed of at least two repetitive units of amino acids, with a polypeptide with trans-sialidase activity of Trypanosoma cruzi fused to the C-terminal region. The vaccine further comprises an aluminum oxide adjuvant that does not inhibit trans-sialidase enzymatic activity of the immunogen portion.

The technical objective of this invention is to strengthen the immuneresponse against protozoan and bacterial antigens, especially toincrease the induction of the cytotoxic T response, essential againstthese antigens. This invention will lead to the development oftherapeutic or prophylactic vaccine formulations for the Chagas disease.

STATE OF THE ART

Trypanosoma cruzi is a protozoan of the Kinetoplastida order,Tryponosomatidae family, distinguished by the presence of a singleflagellum and a single mitochondrion, within which its genome is orderedin a complex and compact network called kinetoplast. It is anintracellular parasite with a life cycle involving vertebrates andinvertebrates.

There are three different forms: Amastigote: spherical or oval, it isthe reproductive form in the interior of the mammal cells, Epimastigote:elongated, with the kinetoplast located anterior to the nucleus, it isthe reproductive form in the digestive tract of invertebrates and inculture media, and Trypomastigote: also elongated, but with thekinetoplast located posterior to the nucleus. Found in the mammals bloodand is their infecting form. This form is not divided.

T. cruzi is divided in two large groups: T. cruzi I and T. cruzi II. Thelatter is divided into five smaller groups: T. cruzi IIa, IIb, IIc, IIdand IIe.

The Chagas disease etiologic agent also known as Americantripanosomiasis is a protozoan intracellular parasite, Trypanosomacruzi. It is transmitted by a hematophagous insect, the Triatomainfestans, which transmits the parasite when the insect defecates on thebite wound as it feeds. On mammals the cycle of T. cruzi cycles betweentrypomastigote stage which circulates in the blood and the amastigotestage which replicates in the cytoplasm of infected host cells(especially on muscles). Chagas disease prevails in most Latin Americancountries including Mexico and Central America, where approximately 18million people are infected with T. cruzi and at least 50.000 childrenand adults die every year from chronic Chagas disease due to lack ofeffective treatments.

The reduvid triatoma, known as vinchuca (from Ecuador to Patagonia),chipo (in Venezuela), pito (in Colombia), and barbeiro (in Brazil) arehematophagous insects, that is to say, blood suckers, that live incracks, holes and dirty areas in houses or cellars in South America andCentral America regions. They become infected after biting an animal orperson that already suffers from the disease. In general, the infectionis spread to human beings when an infected insect deposits feces on aperson's skin while the person is sleeping at night.

The person often rubs the bite accidentally introducing feces in thebite wound, an open cut, eyes or mouth. Animals can also be infected inthe same way and also contract the disease eating the infected insect.The infected person may not present symptoms of the disease until 10 or15 years after being infected; this makes the detection of the diseaseeven more difficult.

More than 90 million people are at risk of infection in endemic areas.In addition, in the endemic areas, 2-5% of fetus carried by infectedmothers are aborted or born with the congenital Chagas disease.

Loss of revenue in terms of productivity lost due to sickness and highmedical costs have an overwhelming effect in the economic growth ofthese countries. The risk of transmission of T. cruzi to non-infectedindividuals through organ transplants and blood transfusions frominfected immigrant donors is very high.

Chemotherapeutical treatments have been partially successful incontrolling T. cruzi infection and Chagas disease. However, the hightoxicity of drugs and poor efficacy of available therapeutics haslimited the use of chemotherapy for treatment of both acute and chronicpatients. Further, drug therapy reduces the severity of disease inchronically infected individuals but cannot reverse the damage alreadydone by parasites.

There are practically no vaccines for the prevention or treatment of theT. cruzi infection. Traditional vaccines constituted of heat-inactivatedparasites, or subcellular fractions of the T. cruzi provide a degree ofprotection for T. cruzi infections (M. Basombrio, Exp. Parasitol. 71:1-8(1990); A. Ruiz et al., Mol. Biochem. Parasitol, 39:117-125. (1990)).However, these vaccines fail to elicit the protective level of immunity,probably due to loss of important epitopes during inactivation and/orthe failure of the antigens to enter the Major HistocompatibilityComplex (MHC) class I pathway of antigen processing and presentation,and to elicit cell mediated immune responses (J. Mónaco. Immunol. Today13:173-179 (1992)). Live attenuated vaccines are capable of entering theMHC class I pathway and might elicit protective immune responses.However, the danger of reversion of the attenuated parasites to virulentstrains if attenuation is not been completed renders these vaccinesimpracticable. A DNA vaccine containing the gene codifying atrans-sialidase has been shown to provide prophylactic protectionagainst T. cruzi infections in mice (F. Costa et al, Vaccine 16:768-774(1998)), but has not been shown to prevent or reverse disease or tostimulate a CD8+ T cell response in animals. In addition, the specificcellular and humoral immune response in BALB/c mice immunized with anexpression genomic library of the T. cruzi was observed (E. Alberti etal., Vaccine 16:608-612 (1998)).

Trans-sialidase is a Trypanosoma cruzi enzyme (agent that causes Chagasdisease) required by this parasite to invade cells of the human host.Given the fact that if the parasite does not invade cells, it will notsurvive in humans, trans-sialidase seems the ideal target for animmunological attack, that is, to develop a vaccine. Therefore, theobjective is a vaccine that as a response when used to immunize, mayproduce antibodies specifically inhibiting the trans-sialidase.

There are several trans-sialidases produced in the trypanosome. Somehave only one region required for the enzymatic activity (thetrans-sialidation which is the transfer of a sugar called sialic acid).Others, in addition to this region have a second region not related totrans-sialidation but that is very immunogenic (it generates antibodiesin the host). This second region is called SAPA(Shed-acude-phase-antigen) and is formed by repetitive units of aminoacids.

The gens (nucleic acids, DNA, formed by units called nucleotides orbases, a region of the DNA that codifyings a protein as in this casetrans-sialidase, is called gen) codifying the region with enzymaticactivity and the SAPA region have been identified.

One of the T. cruzi molecules described as essential for the host cellinvasion is the sialic acid (Schenkman S. Et al. Cell 65, 1117-1126,1991; Schenkman, S. et al. Ann. Rev. Microbiol. 48, 499-523, 1994;Schenkman, S. and Eichinger, D. Parasitology Today 9, 218-222, 1993).Since trypanosome is unable to synthesize sialic acid (Schauer, R. y etal. Z. Physiol. Chem. 364: 1053-1057, 1983), it must obtain it frommolecules containing sialic acid present in the environment. Thisprocess is accomplished using a unique enzyme called trans-sialidase(Previato, J. O. et. al., Mol. Biochem. Parasitol. 16:8596, 1985 YZingales, B., et al., Mol. Biochem. Parasitol. 26, 135-144, 1987).Trans-sialidase is capable of transferring sialic acid from sialydatedmolecules present in the environment, such as some molecules found inthe blood of the infected host, to molecules present on the trypanosomesurface.

Trypanosome molecules that can be sialylated are those called mucins(Ruiz, R. C., et al. Parasite Immunol. 15,121-12, 1993; M. B. Reyes, etal. Gene 140, 139-140, 1994; J. M. Di Noia et al. J. Biol. Chem. 270,24146-24149, 1995; J. M. Di Noia, et al. J. Biol. Chem. 271,32078-32083, 1996). Once sialylated, mucins are the molecules thatinteract with the surface of the human cell to be invaded, facilitatingthe infection process (Ruiz, R. C., et al. Parasite Immunol. 15, 121-12,1993). Other groups have demonstrated that if the parasite does notexpress trans-sialidase, it cannot infect cells in the same way asparasites containing trans-sialidase do (Pereira et al., Infect. Immum.64, 38843892, 1996). Therefore, immunization with trans-sialidase madein such a way that generates antibodies to inhibit the enzyme,constitutes a useful tool to obtain a vaccine against this parasite.Recently it has been demonstrated that the immune response againsttrans-sialidase is a factor that helps prevent death of the host causedby the parasite ((Chuenkova, M. y Pereira M. E. A., J. Exp. Med. 181,1693-1703, 1995).

Trypanosoma cruzi has two types of trans-sialidases. One type containsonly the amino acids required for the activity of trans-sialidase(Briones, M. R. S. et al. Mol. Biochem. Parasitol. 70: 9-17, 1995) andwas used in the application WO 9318787 with the purpose to synthesizecarbohydrates due to its enzymatic activity and was proposed in secondplace as an immunogen in the same patent application. A second group oftrans-sialidases contains, in addition to these sequences, a series ofamino acid repetitions in the terminal carboxyl region called SAPA (C.Ibáñez, et al. Mol. Biochem. Parasitol. 30: 27-34, 1988; J. L.Affranchino, et al. Mol. Biochem. Parasitol 34: 221228, 1989; Cazzulo,J. J. and Frasch, A. C. C. FASEB J. 6, 3259326, 1992). This secondregion is highly antigenic during a natural infection in humans (M. B.Reyes, et al., Proc. Natl. Acad. Sci. USA 87:2846-2850, 1990).

The broader investigations for vaccines have been focused on attempts todevelop prophylactic protein vaccines against the infection of T. cruzi,but have been carried out with little success. The development ofvaccines of subunits composed by defined antigens capable of inducingstrong humoral and class 1 T cell responses and of reducing the parasiteburden, has been hindered due to lack of knowledge of biology of thethree developing stages of the T. cruzi, the absence of sufficientinformation of the sequence on gens expressed in the contagious andintracellular stages, and the scientific view that chronic disease isnot related to persistent parasite infection but it is the result of aparasite-induced autoimmune response.

SUMMARY OF THE INVENTION

The first object of the present invention is a vaccine against Chagasdisease, capable of stimulating the immune response against thetrans-sialidase virulence factor of the parasite Trypanosoma cruzi, thevaccine is distinguished by the fact that it comprises a multicomponentvaccine for the Chagas disease (American tripanosomiasis) comprising:(a) an immunogenic portion formed by one or more recombinant orsynthetic polypeptides or fractions thereof and (b) one or morepolynucleotides comprising the regions that codifies one or moreimmunogenic polypeptides, both portions for the Trypanosoma cruziderivates (i.e., T. cruzi and or a conserved region common to many ofthem) where the vaccine administration is a protection against theparasite infection, eliminates it or reduces the clinical consequencesof the infection.

Another objective of the present invention is a monocomponent vaccineagainst the Chagas disease that comprises at least one componentselected among an immunogenic portion formed by one or more recombinantor synthetic polypeptides or fractions thereof, and a group ofpolynucleotides including the regions that codifying one or moreimmunogenic polypeptides derived from Trypanosoma cruzi (i.e. a T. cruziand/or a conserved region common to many of them) where the immunogenicportion or the polynucleotides group stimulates an antibody response, ofCD4+ Th1 biased T cells or CD8+ T cells against the Trypanosoma cruzi.

This invention also includes the pharmaceutical compositions containingthe multicomponent and monocomponent vaccines, the procedures for theobtention of the immunogen portion of said vaccines and the nucleic acidused in said procedure.

FIGURES DESCRIPTION

FIG. 1: SEQ ID NO: 1. Sequence of the nucleotides of the gene regioncodifying the Trypanosoma cruzi protein having trans-sialidase activity.Letters represent four bases (molecules) that constitute de DNA(deoxyribonucleic acid). A: adenine, T: tymine, C: cytokine, G: guanineEvery three bases (bases triplet) codifies an amino acid (molecular unitfaulted by the protein) that in this case is trans-sialidase. The ATGindicated in low case is the first amino acid of the trans-sialidase(methionine amino acid). The last TGA triplet is the one used by thecell to indicate where the protein ends (termination triplet).

FIG. 2: SEQ ID NO: 2. Amino acid sequences of the region codified by thegene of SEQ ID NO: 1 showed in FIG. 1 and that corresponds to the partof the protein having trans-sialidase activity. Each letter indicates anamino acid according to the universally accepted code.

FIG. 3: SEQ ID NO: 3. Base sequence codifying the region of repetitiveunits of amino acids called SAPA. Remaining instructions same as SEQ IDNO: 1 shown in FIG. 1.

FIG. 4 SEQ ID NO: 4. Amino acids sequence codifying according to thebase sequence of SEQ ID NO: 3 indicated in FIG. 3 and corresponding toSAPA protein.

FIG. 5: SEQ ID NO: 5. Nucleotides (upper line) and amino acid (lowerline) sequence corresponding to the gene and the protein respectively,resulting from the union of trans-sialidase and SAPA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an efficient vaccine for treating oravoiding the infection of a mammal by Trypanosoma cruzi derivatives(i.e. T. cruzi and/or a conserved region common to many of them). In apreferred embodiment, the vaccine is effective against infection and/orillness caused by T. cruzi. The multicomponent vaccine of this inventionis for the Chagas disease (American tripanosomiasis) comprising: (a) animmunogenic portion formed by one or more recombinant or syntheticpolypeptides, or fractions thereof, and (b) one or more polynucleotidesincluding the regions that codifying one or more immunogenicpolypeptides, both portions for the Trypanosoma cruzi derivates (i.e.,T. cruzi and or a conserved region common to many of them) where thevaccine administration is a protection against the parasite infection,eliminates it or reduces the clinical consequences of the infection. Onepolynucleotide vaccine contains one or more polynucleotides thatcomprise the regions codifying one or more immunogenic polypeptidesderived from T. cruzi. In a similar way, a polypeptide vaccine containsone or more immunogenic polypeptides derived from T. cruzi.

Another objective of this invention is a monocomponent vaccine againstChagas disease comprising at least one component selected between oneimmunogenic portion is selected from one or more recombinant orsynthetic polypeptides, or fractions thereof, and a group ofpolynucleotides that cover the regions that codifying one or moreimmunogenic polypeptides derived from the Trypanosoma cruzi (i.e., of aT. cruzi and/or a conserved region common to many of them) where theimmunogenic portion stimulates an antibody response of CD4+ Th1 biased Tcells or CD8+ T cells against Trypanosoma cruzi.

The “immunogenic portion” of the vaccine may comprise one or morepolypeptides, the structure of which includes a C-terminal region whichconsists of at least two repetitive units; each of those repetitiveunits shows at least 60% homology to the following amino acid sequence:AHSTPSTPVDSS (SEQ ID NO: 6) and a polypeptide with trans-sialidaseactivity is fused to the C-terminal region. It may also comprise anadjuvant which does not destroy the trans-sialidase enzymatic activityof the immunogen portion, preferably aluminum oxide. The portion maycomprise between 10 and 16 repetitive units in the C-terminal region,preferably 13 units.

The immunogenic portion may be obtained from the Trypanosoma cruzitrypomastigotes (i. e. from a T. cruzi and/or a conserved region commonto many of them).

This invention relates to a vaccine that may be a recombinantbiomolecule formed by the fusion of that region which consists ofrepetitive units of amino acids and the polypeptide with trans-sialidaseactivity and/or the polypeptide with cysteine proteinase activity,and/or the Paraflagelar Rod Proteins (PFR).

The multicomponent vaccine of this invention preferably stimulates anantibody response or an immune response transmitted through cells, orboth responses, in the mammal to which the vaccine will be administered.The vaccine preferably stimulated a response of CD4+ Th1 biased T cellsor CD8+ T cells. Preferably in the case of a monocomponent vaccine, thevaccine stimulates the antibody response, a response of CD4+ Th1 biasedT cells or a response of the CD8+ T cells. A form of especiallypreparing the vaccine of this invention includes a nucleotide comprisingthe regions codifying a cytokine, to provide the additional stimulationto the mammal immune system. In a preferred embodiment, the preparationof the vaccine of this invention includes an immunogenic polypeptidewhich contains a sequence of membrane displacement, to facilitate theintroduction of the polypeptide in the mammal's cell and the subsequentstimulation of the immune response transmitted through cells.

The immunogen of this invention may be selected from the TSA-1, ASP-1,ASP-2, hemolysin and Lyt1 proteins.

The multicomponent vaccine of this invention may comprise a plurality ofpolynucleotides that comprise the regions codifying one or moreimmunogenic polynucleotides derived from the T. cruzi (of a T. cruziand/or a conserved region common to many of them) and at least one ormore polynucleotides comprising the regions codifying the cytokines thatmay be selected among interleukin 12 (IL-12), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin 6 (IL-6), interleukin 18(IL-18), γ-interferon, α,β-interferons and chemokines; the IL-12 andGM-CSF cytokines are especially preferred.

The pharmaceutical compositions that contained the recombinant orsynthetic polypeptides, or fractions of the immunogenic portion and thepolynucleotides including the regions which codifying one or moreimmunogenic polypeptides derived from the T. cruzi, together with apharmaceutical carrier are also the object of this invention.

In another embodiment, this invention also provides a vaccine ofmultiple polynucleotide components. It is prepared by inserting two ormore nucleotides comprising the regions which codifying one or moreimmunogenic polypeptides derived from the T. cruzi in two or morepolynucleotide vectors, later combining the polynucleotide vectors toyield a polynucleotide vaccine.

Alternatively, this invention related vaccine may be prophylacticallyadministered to a mammal before the T. cruzi infection. In a preferredembodiment the vaccine application must be efficient to prevent thesubsequent infection of the mammal with T. cruzi. In another embodimentthe vaccine administration is efficient to prevent the development ofthe chronic debilitating disease in a mammal after the subsequentinfection with T. cruzi. In other embodiment, the vaccine application isefficient to prevent mammal's death after the subsequent infection withT. cruzi.

In another embodiment, the invention includes a method for identify theimmunogenic polypeptides of T. cruzi from a T. cruzi library, to be usedin a polynucleotide vaccine. In a preferred embodiment, the procedureutilizes the expression library immunization (ELI) in mice to identifythe T. cruzi polypeptides that elicit an immune response in a mammaleffective to prevent the death, arrest or delay the progression ofdisease in the mammal that has been infected with T. cruzi. The methodis preferably used to identify the immunogenic polypeptides derived fromthe T. cruzi and from the BALB/c or B6 mice which have been immunized.

In another embodiment the method involves the following:

-   (a) preparing a DNA microarray comprising open reading frame of T.    cruzi genes;-   (b) preparing a first probe comprising Cy3-labeled    trypomastigote-derived T. cruzi cDNA;-   (c) preparing a second probe comprising Cy5-labeled    amastigote-derived cDNA;-   (d) cohybridizing the first and second probes to the microarray to    identify at least one gene whose expression is the T. cruzi during    the intracellular amastigote stage of the infectious cycle, where    the gene codifies a candidate immunogenic T. cruzi polypeptide; and-   (e) immunizing mice with the gene to determine whether the gene    codifies a T. cruzi polypeptide that elicits an immune response in a    mammal effective to prevent the death of the mammal or to arrest or    delay the progression of disease in the mammal associated with    infection of the mammal by T. cruzi.

A form of preparing the immunogen used in the multicomponent ormonocomponent vaccines of this invention comprises the following steps:a) bindung the codifying nucleotidic sequences for the immunogenicpeptide/s to a vector capable of expressing such sequence, b) linkingthe codifying sequence obtained in the previous step to a vector capableof expressing such sequence, c) transforming a host capable ofexpressing the codifying sequence of the immunogen, d) growing thetransformed bacteria obtained in the previous step in an appropriateculture medium, e) isolating and purifying the immunogen obtained in theprevious step.

Stage b) vector may be pET22b+. The hosts are eukaryote cells, bacteria,especially Escherichia coli BL26DE3, and yeasts. The culture medium ofstage d) may be an L-Broth medium.

Another form of preparing the immunogen used in the multicomponent ormonocomponent vaccines of this invention comprises the following stages:a) growing trypomastigote form of Trypanosoma cruzi in an adequateculture medium, b) obtaining the supernatant where the trypomastigoteforms have grown by centrifugation at 5000 rpm during 10 minutes, c)filtrating the supernatant obtained and passage through an affinitycolumn containing immunoglobulins that recognize the repetitions of thecarboxymethyl terminal sequence of the immunogen, d) eluting increasingthe pH to detach the protein.

Peptides used in the procedures of this invention may be obtained bychemical synthesis.

Another object of this invention comprises a nucleic acid used in theprocedures previously explained because it essentially comprises asequence codifying said immunogen.

Over 100 natural mammalian hosts are known for T. cruzi-hosts, and T.cruzi can be transmitted to a human from another animal host. Anymammalian host can be immunized according to this invention. Vaccineadministration in this application includes humans, domestic animalssuch as dogs and cats, rodents and wildlife animals. Preferably, themammal that is immunized is a dog, a cat or a human.

Polynucleotide Vaccine

The polynucleotide vaccine of the invention includes at least one,preferably at least two, nucleotides coding regions, each coding regiongencoding an immunogenic polypeptide component from T. cruzi. When itcontains two or more nucleotide coding regions, the polynucleotidevaccine is referred to herein as multicomponent vaccine. It isconvenient to minimize the number of different immunogenic polypeptidesencoded by the nucleotide coding regions; however, considering that apolynucleotide vaccine generates the highest level of protection, itwill codifie 10 or more immunogenic polypeptides. The polynucleotidevaccine contains DNA, RNA, a modified nucleic acid, or any combinationthereof. Preferably, the vaccine comprise one or more expression orcloning vectors; more preferably, the vaccine comprises a plurality ofexpression vectors capable of autonomous expression of a nucleotidecoding region in a mammalian cell to produce at least one or moreimmunogenic polypeptides or cytokine. An expression vector preferablyincludes a eukaryote promoter sequence, more preferably the nucleotidesequence of a strong eukaryotic promoter operatibly linked to one ormore coding regions. A promoter is a DNA fragment which acts as aregulatory signal and binds the RNA polymerase in a cell to initiate thetranscription of a downstream (direction 3′) coding sequence; thetranscription is the formation of an RNA chain in accordance with thegenetic information contained in the DNA. A promoter is operatiblylinked to a nucleic acid sequence, or may be used to control or regulatethe transcription of that nucleic acid sequence. This invention is notlimited to a specific eukaryotic promoter but extends to the widevariety known; preferably, the expression vector includes a CMV or RSVpromoter. The promoter used preferably remains as constitutive promoter.

A vector useful in the present invention can be circular or linear,single-stranded or double-stranded and can be a plasmid, cosmid oreposome but is preferably a plasmid. In a preferred embodiment, eachnucleotide coding region (whether it codifies an immunogenic polypeptideor a cytokine) is on a separate vector; however, it is to be understoodthat one or more regions can be present on a single vector, and theseregions can be under the control of single or multiple promoters.

Nucleotidic sequences codifying cytokines may be added to thepolynucleotide vaccine, such as a granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin 12 (IL-12) and theco-stimulating molecules, such as B7-1, B7-2, CD40. Cytokines may beused in several combinations to fine tune the response of the animal'simmune system, including both antibody and cytotoxic T lymphocyteresponses, achieving a specific level of response needed to control oreliminate the T. cruzi infection. The polynucleotide vaccine may includea fusion product containing an antigenic polypeptide and a molecule,such as CTLA-4, which directs the fusion product to the cells withantigen presence within the host.

Plasmids and other delivery systems are prepared using well knowntechniques in molecular biology. This invention may include proceduresto prepare and use the polynucleotide vaccine.

Polypeptide Vaccine

The polypeptide vaccine of this invention includes at leastone—preferably at least two—immunogenic polypeptides from T. cruzi(recombinant or synthetic, or fractions thereof). As with thepolynucleotide vaccine, it is desirable to minimize the number ofdifferent immunogenic polypeptides supplied in the vaccine; however, itis nonetheless contemplated that a polypeptide vaccine that generatesthe highest level of protection will contain IQ or more numerousimmunogenic polypeptides.

Because a CD8+ T cells response cannot normally be directly triggered bythe administration of a conventional protein subunit vaccine, theimmunogenic polypeptides contained in the polypeptide vaccine includepreferably include one or more membrane transporting sequences (MTS)fused to their N-terminus or C-terminus or both. A membrane transportingsequence allows for transport of the immunogenic polypeptide across alipid bilayer, allowing it to be delivered to the inside of a mammaliancell.

Cytokines

The Cytokines are proteins which regulate the function of the cells thatproduce them or other cellular types. They are the agents responsiblefor intercellular communication; they induce the activation of membranespecific receptors, proliferation functions and cellulardifferentiation, chemiotaxis, growth and modulation of immunoglobulinsecretion. They are basically produced by activated lymphocytes andmacrophages, although they may also be produced by polynuclearleukocytes, endotelial cells, epitelial cells and conjunctive tissuecells. Depending on the cell that produces them, they are calledlymphokines (liymphocyte), monocynes (monocyte) or interleukines(hematopoietic cells). Their basic action is the regulation of theinflammation mechanism. There are pro-inflammatory and anti-inflammatorycytokines.

Preferably, the polynucleotide vaccine includes at least one nucleotidecoding the region codifying a cytokine. Preferred cytokines includeinterleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor(GM-CSF), interleukin 6 (IL-6), interleukin 18 (IL-18), γ-interferon,α,β-interferons, and chemokines. Especially preferred cytokines includeIL-12 and GM-CSF.

Pharmaceutical Compositions

The polynucleotide and polypeptide vaccines of the invention are readilyformulated as pharmaceutical compositions for veterinary or human use.The pharmaceutical composition optionally includes excipients ordiluents that are pharmaceutically acceptable such as those that werecompatible with the genetic material. The term “pharmaceuticallyacceptable carrier” refers to a carrier that is acceptable in the sensethat it is compatible with the various components of a composition andthat does not negatively affect its therapeutical behavior. Suitableexcipients are, for example, water, saline solution, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, salts, and/orcoadjuvants that enhance the effectiveness of the stimulatingcomposition of an immune response. In this invention some procedures toprepare and use those pharmaceutical compositions are also included.

Administration of a Combination of a Polynucleotide Vaccine and of thePolypeptide Vaccine

This invention comprises the administration of a polynucleotide vaccineand of a polypeptide vaccine to a mammal in a serial protocol. Forexample, a plasmid-based DNA vaccine may be administered to the primaryimmune system of a mammal, followed by one or more administrations of apolypeptide vaccine or of a viral vaccine (for example, vector of thevaccine polypeptide that carries the genes that codifies the immunogenicpolypeptides and, optionally, the cytokines) to stimulate the mammal'simmune system. The order of administration of the various types ofvaccines and the nature of the vaccines administered, in any dosage (forexample the polypeptide vaccine, plasmid vaccine, viral vector vaccine)may be easily determined by an expert to provoke the most efficientimmune response in the mammal.

Definitions of Certain Words and Expressions

“Chagas disease” refers to different clinical expressions in patientsinfected by different varieties of the T. cruzi, related at the sametime to the chronic immunological impact on various white tissues.Consequently, vaccines presently known, either from monkeys ormulticomponents from frequently available substances of some T. cruzitype, are partial formulas to undertake a potentially effectivetreatment in every geographical area where this disease develops, andagainst every intra o extra-cellular form of the parasite. In addition,the immunogenicity of such common components may vary over time and/orwith changes in the host health condition, and therefore, a universalcontinuous protection is not guaranteed.

“T Lymphocyte” refers to cells (white blood cells) in charge ofcoordinating the cell-mediated immune response, as well as cooperationfunctions for the development of every kind of immunological responses,including antibodies response by B lymphocytes. T lymphocytes may bedifferentiated from B lymphocytes and killer cells due to the appearanceof a special receptor in the surface of the membrane, named T-cellsReceptor (TCR). T comes from thymus, the most important organ for thedifferentiation of these cells from mother cells of the lymphaticsystem.

“CD4+ T cell” refers to T lymphocytes responsible for coordinatingcell-mediated immune response, as well as cooperation functions for thedevelopment of every kind of immunological response, includingantibodies response by B lymphocytes.

“CD8+ T cell” refers to Cytotoxic T Lymphocytes T (CTL); they areincluded in the T lymphocytes line responsible for the effector functionof cell immunity. They neutralize cells infected by intracellularmicroorganisms, by attacking directly infected cells, injecting toxicenzymes and destroying them. They are currently named CD8+, due to thepresence of the CD8 membrane receptor.

CTLs are essentially able to lyse cells when stimulated adequately,especially by antigens expressed on MHC class I. Very specific in theirlethal functions, they have the ability to destroy a target cell withoutaffecting surrounding uninfected cells. The cell CTL-mediateddestruction process comprises:

-   Recognition of foreign antigen and formation of a stable conjugate    with membrane receptors; MHC-I, TCR, CD8, ICAM-1, LFA-1, among other    receptors and co-stimulators;-   to Activation of CTL cell by means of cell interrelations mediated    by membrane proteins and transduction of intracellular signs;-   Cell lysis compelling to changes in target's cell and exocytosis of    poisoning granules containing principally perforin y granzine;-   Apoptosis of the target cell including the mediation of molecules    inducing cell death, like Fas and its ligand.

Due to the high toxicity of these lymphocytes, and in order to preventthe unnecessary risk of continuous circulation, inactive or virgincytotoxic cells require two types of triggering signals:

-   Binding of T cells receptor in the membrane of the cytotoxic cell to    Major Histocompatibility Complex (MHC-I) type I on the surface of    cells presenting antigen (CPA),-   Cells infected by microorganisms, preferably intracellular ones    (like viruses), show remnants of the microorganism on their surface    in the context of a MHC-I molecule. The infected cell shows antigens    foreign to a CPA that processes infecting information to CTLs,-   Co-stimulation at high concentrations derived from the binding    between CD40 molecule appearing on membranes of dendritic cells and    their ligand (CD40L) of cytotoxic cells. It is a redundant    interaction, that is to say, the more contacts between CD40 and its    ligand, the greater is the production of ligands by the CTL, turning    triggering signal stronger and more definitive.-   In infected tissues, CTLs recognizing the antigen involved are    activated and retained in the infection area, and they carry out    their effector activity. Those CTLs not recognizing the antigen    involved return to circulation.

The “Immunogenic polypeptide” used in the vaccine against T. cruziaccording to this invention refers to the one that can be expressed byT. cruzi in extracellular stage (trypomastigote), in intracellular stage(amastigote), or during both stages of the life cycle. Preferably, theimmunogenic polypeptide is expressed by the T. cruzi amastigote in earlystage of infection, in approximately 24 hours after initial infection.

A type of polypeptides exemplifying immunogenic polypeptides accordingto the invention is from the family of the trans-sialidase proteins,such as TSA-1 (T. cruzi Peru; D. Fouts et. al. Mol. Biochem. Parasitol.46:189-200 (1991); GenBank. Acc. Number M58466), ASP-1 (T. cruzi elBrazil; M. Santos et al. Mol. Biochem. Parasitol. 86:1-11 (1997):GenBank. Acc. Number U74494)) and ASP-2 (T. cruzi el Brazil; H. Low etal. Mol. Biochem. Parasitol. 88:137-149 (1997); GenBank. Acc. NumberU77951).

Advantages of this Invention

This invention is the most complete multicomponent or monocomponentvaccine that may constitute varieties of the same component of differentT. cruzi, especially of those portions conserved and/or with theaddition of targets different among them, for example taking as a targetone or several trans-sialidases, fragellar proteins or cysteineproteases from one or more varieties of T. cruzi.

Chagas is a disease requiring incentives and protection for thedevelopment and distribution investments; therefore there is no room fora competitive, multi-optional market, as if an Argentine vaccine, aBrazilian vaccine, a Peruvian vaccine, etc. co-existed; in practice,presently known vaccines could not be developed without leavingunprotected an important part of the population affected. Consequentlythe invention involves the maximum immunogenicity combination facing thevariety of clinical expressions (that is, a “monoshot-continental”vaccine).

A formula is proposed to be used against all varieties and clinicalexpressions. Formulas with such wide range of multicomponents have notbeen described up to this date.

Advantages of a Genetic Vaccine

Choosing the administration of the polynucleotide as an immunizationtechnique offers an excellent advantage over the different systems thatsupply other vaccines or antigen delivery systems. Vaccines containinggenetic material are preferred over traditional vaccines because vectorsare easy to construct and produce, the potential for the modification ofmutagenesis sequences directed to enhance antigenic potency ofindividual epitopes, or to abolish epitopes that might trigger anunwanted response. In the case of DNA vaccines, DNA stability, the lackof the risks associated to live and attenuated vaccines, their abilityto induce humoral immunity and cell transmitted immunity, and,particularly, CD8+ T cell responses, and persistence of the immunityresponses.

It has also been demonstrated their ability to improve immunity responseby the co-administration of genes codifying cytokines.

TABLE 1 TS activity in serum following intravenous injection oftrans-sialidase protein (TS) or SAPA-bound trans-sialidase (TS-SAPA).Hours post-inoculation 0.5 16 26 110 TS* 100% 3% 0% 0% TS-SAPA* 100% 60%50% 5% Values are averages obtained from three independent animals pergroup. Measuring was carried out on serum samples collected from eachmouse at 30 minutes, and 16, 26 and 110 hours following inoculation. Themouse strain used was C3H. *Values expressed in % activity of remainingtrans-sialidase considering as 100% the values obtained 0.5 h followingTS immunization (for TS values) or with TS-SAPA (for TS-SAPA values)respectively.

If instead of injecting a protein with trans-sialidase activity, a mouseis intravenously injected a protein containing the region withtrans-sialidase activity bound to SAPA protein (TS-SAPA), resultsobtained are different (Table 1). When samples are collected atdifferent times following intravenous injection, trans-sialidaseactivity is detectable up to 5 days after the injection. These resultsshow that SAPA protein, when bound to another protein liketrans-sialidase, is conferred a new property: the property ofstabilizing it in blood, maintaining this enzymatic activity incirculation for a longer time.

Results similar to those described above may be obtained when otherroutes of administration, such as, intramuscular, intraperitoneal,subcutaneous or any other triggering an antibody response in theimmunized host are used instead of the intravenous route ofadministration. Similar results can be obtained as well when some of thealready known immunizing adjuvants are used, such as aluminum oxide orothers not destroying the enzyme, as detailed below. An adjuvant iseither an oily or aqueous substance, or a substance with anotherstructure that injected jointly with the immunogen—in this case arecombinant protein like trans-sialidase/SAPA-contributes to increaseimmunogenicity, that is, increases the immune system response related toantibodies production or cell response. A cell response is a type ofresponse by the immunologic system where basically the cellscontributing to control an infectious agent proliferate.

Another result obtained from these experiments is that a responseagainst trans-sialidase inhibiting its activity is only obtained whenthis enzyme is used as an immunogen in its active form, that is, with aconformation that retains its enzymatic activity.

TABLE 2 Inhibiting activity of the trans-sialidase in serum of animalsimmunized with trans-sialidase, with repetitions (TS-SAPA), withFreund's adjuvant (AF), with aluminum oxide (Al₂0₃) or with proteindenaturalized by heat at 80° C. (80° C.) Immunization was carried out byintraperitoneal route, in all cases with 65 nanogrames for the firstdose (day 0) and with 13 nanogrames for the second dose on day 38.Trans-sialidase inhibition measure was carried out as described for theexperiments previous to day 1 and on day 45 after the first immunization(day 0). The measure is expressed in % of the inhibition obtained in thepresence of serum of non immunized mice, the inhibition values of whichare considered 0. The values are the average obtained from fourimmunized mice independently for each condition. Days after immunization1 45 TS-SAPA in AF 0% 0% TS-SAPA in Al₂0₃ 0% 90% TS-SAPA at 80° C. 0% 0%

Another result obtained as a consequence of these experiments is thatonly a response is obtained against the trans-sialidase that inhibitsits activity when this enzyme is used as immunogen in its activeformulation, that is, with such a conformation that it keeps itsenzymatic activity. Table 2 shows that when the adjuvant used toimmunize mice is aluminum oxide—which does not alter the enzymaticactivity of trans-sialidase—antibodies which inhibit it are generated.However, if Freund is used as adjuvant—which destroys the enzymaticactivity of trans-sialidase, no antibodies inhibiting trans-sialidasewill be generated in the mouse. Likewise, heat-inactivatedtrans-sialidase at 80° C. for 3 minutes so as to avoid protein frommaintaining it enzymatic activity, preventing it from being stimulatedby the formation of neutralizing antibodies.

Results similar to those described above may be obtained when otherroutes of administration, such as intramuscular, intraperitoneal,subcutaneous or any other triggering an antibody response in theimmunized host are used instead of the intravenous route ofadministration.

Furthermore, similar results may be obtained when some of the adjuvantsknown to immunize, such as aluminum oxide or others which do not destroythe enzyme, as mentioned before. An adjuvant is oily, water, or otherstructure substance, that injected together with the immunogen, in thiscase a recombinant protein, such as trans-sialidase/SAPA, contributes toincrease its immunogenicity, that is, it increases the response of theimmune system as regards antibodies production or cell response. Cellresponse is a type of response of the immunologic system whereproliferation is basically of cells contributing to control aninfectious agent.

There is no doubt that once this invention is put into practice, somemodifications may be introduced regarding construction and shapedetails; this will not imply straying away from the fundamentals thatare clearly expressed in the claims that follow.

I claim:
 1. A monocomponent vaccine effective to moderate the clinicalconsequences of Chagas disease, said vaccine comprising, as activeingredient, an immunogenic component comprising a polynucleotide, saidnucleotide encoding one or more polypeptide(s) each having a C-terminalregion composed of at least two repetitive units of amino acids and aeukaryotic promoter operably linked to one or more coding regions forsaid polypeptide(s), wherein each repetitive unit of amino acids showsthe following amino acid sequence: AHSTPSTPVDSS (SEQ ID NO: 6) andwherein a polypeptide with trans-sialidase activity of Trypanosoma cruziis fused to the C-terminal region of each of said polypeptide(s),wherein the polypeptide with trans-sialidase activity of Trypanosomacruzi has the amino acid sequence of SEQ ID NO. 2 and is encoded by thenucleotide sequence of SEQ ID NO. 1, and wherein the vaccine furthercomprises an adjuvant that does not inhibit trans-sialidase enzymaticactivity of the immunogen portion, wherein the adjuvant is aluminumoxide.
 2. The monocomponent vaccine of claim 1, wherein the C-terminalregion of the immunogenic component is encoded by a polynucleotidehaving the nucleotide sequence of SEQ ID NO.
 3. 3. The monocomponentvaccine of claim 1, further comprising one or more polynucleotides thatcomprise the regions which encode cytokines.
 4. The monocomponentvaccine of claim 3, wherein the cytokine is selected from interleukin 12(IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin 6 (IL-6), interleukin 18 (IL-18), γ-interferon,α,β-interferons and chemokines.
 5. The monocomponent vaccine of claim 4,where the cytokine is selected from the IL-12 and GM-CSF cytokines. 6.The monocomponent vaccine defined in claim 1, wherein the immunogeniccomponent is selected from between 10 and 16 repetitive units in theC-terminal region.
 7. The monocomponent vaccine defined in claim 1,comprising a recombinant biomolecule formed by the fusion of that regionwhich comprises repetitive units of amino acids and at least one of (i)the polypeptide with trans-sialidase activity, (ii) the polypeptide withcysteine proteinase activity, and (iii) the paraflagellar rod protein(PFR).
 8. The monocomponent vaccine defined in claim 1, wherein theimmunogen of the immunogenic component is obtained from Trypanosomacruzi trypomastigotes.
 9. The monocomponent vaccine defined in claim 1,wherein the immunogen of the immunogenic component is obtained fromTrypanosoma cruzi amastigotes.
 10. The monocomponent vaccine defined inclaim 1, which stimulates at least one immune response selected from anantibody response and an immune response by cell mediation.
 11. Themonocomponent vaccine defined in claim 10, which stimulates at least oneresponse of CD4+Th1 biased T cells and CD8+ T cells.
 12. Themonocomponent vaccine defined in claim 11, which stimulates at least oneresponse CD8+ T cells.
 13. The monocomponent vaccine defined in claim 1,wherein the polypeptide is selected from the TSA-1, ASP-1, ASP-2,hemolysin and Lyt1 proteins.
 14. The monocomponent vaccine of claim 1,wherein said vaccine is a therapeutic vaccine.
 15. The monocomponentvaccine defined in claim 1, wherein said vaccine is a prophylacticvaccine.
 16. The monocomponent vaccine defined in claim 1, wherein theC-terminal region of the immunogenic component is a polypeptide havingthe amino acid sequence of SEQ ID NO.
 4. 17. An isolated polynucleotideencoding one or more polypeptide(s) each having a C-tejininal regioncomposed of at least two repetitive units of amino acids and aeukaryotic promoter operably linked to one or more coding regions forsaid polypeptide(s), wherein each repetitive unit of amino acids showsthe following amino acid sequence: AHSTPSTPVDSS (SEQ ID NO: 6) andwherein a polypeptide with trans-sialidase activity of Trypanosoma cruziis fused to the C-terminal region of each of said polypeptide(s),wherein the polypeptide with trans-sialidase activity of Trypanosomacruzi has the amino acid sequence of SEQ ID NO. 2 and is encoded by thenucleotide sequence of SEQ ID NO. 1, and wherein the vaccine furthercomprises an adjuvant that does not inhibit trans-sialidase enzymaticactivity of the immunogen portion, wherein the adjuvant is aluminumoxide.
 18. The isolated polynucleotide according to claim 17, whereinthe polypeptide with trans-sialidase activity of Trypanosoma cruzi hasthe amino acid sequence of SEQ ID NO. 2 and is encoded by the nucleotidesequence of SEQ ID NO.
 1. 19. A plasmid, cosmid or eposome comprising anisolated polynucleotide encoding one or more polypeptide(s) each havinga C-terminal region composed of at least two repetitive units of aminoacids and a eukaryotic promoter operably linked to one or more codingregions for said polypeptide(s), wherein each repetitive unit of aminoacids shows the following amino acid sequence: AHSTPSTPVDSS (SEQ ID NO:6) and wherein a polypeptide with trans-sialidase activity ofTrypanosoma cruzi is fused to the C-terminal region of each of saidpolypeptide(s), wherein the polypeptide with trans-sialidase activity ofTrypanosoma cruzi has the amino acid sequence of SEQ ID NO. 2 and isencoded by the nucleotide sequence of SEQ ID NO. 1, and wherein thevaccine further comprises an adjuvant that does not inhibittrans-sialidase enzymatic activity of the immunogen portion, wherein theadjuvant is aluminum oxide.